Novel vectors for gene transfer into human ES cells

Human embryonic stem cells have a great potential for medical therapeutics. However, the genes required for altering the fate of these cells to differentiate into a particular tissue or cell type is not well understood. The ability to efficiently transfer genes or silence genes in ES cells would be of great benefit for two reasons: 1) A combination of gene therapy and ES stem cells will likely broaden the therapeutic potential of these cells, and 2) the ability to alter gene expression will provide important tools for unraveling the genetic programs required for targeted differentiation into a specific tissue or cell-type. Viral gene transfer vectors are derived from viruses. The viral genes are removed and replaced with a therapeutic gene or a gene under biological study. Thus, the viral shell is used to transfer the desired gene into cells. Although there has been some success, current gene transfer vectors do not work efficiently in ES cells. To make this new gene transfer vector, we will use the adenoassociated virus (AAV). This virus is not associated with any known disease and has been used in a number of human clinical trials. Another advantage of vectors based on these viruses is that they can be used to overproduce a gene product, turn off a gene product, and even target a mutation in a specified gene. We plan to use a recent strategy developed in our laboratory to select for AAV viruses that are very efficient at infecting human ES cells. Once these viruses are identified, as a proof-of-concept, we will make gene transfer vectors and test them in new human ES cells for their ability to over-express and turn off genes that we believe are important in early human ES cell differentiation.

Statement of Benefit to California:

California is leading the way in stem cell research. The ability to transfer nucleic acids into stem cells will open up an additional therapeutic window as well as allow important tools for helping define the genetic programs required for stem cell differentiation into a myriad of different cell types. The intellectual property surrounding the vectors that we have selected and characterized by the same approach for liver transduction have been protected by a patent application submitted on behalf of {REDACTED}. Like all of our reagents and discoveries, the ES cell specific gene transfer vectors developed in our laboratory will be made available to all academic researchers around the world. We will offer these vectors to corporate entities per the rules and regulations dictated by the California State Stem Cell Institute and {REDACTED}. We should note that even prior to publication, several vectors isolated and characterized for liver gene transfer by our laboratory have been sent to commercial entities for evaluation and possible use in both research and clinical trial development. We believe that our technology will have wide-spread use by stem cell researchers in California as well as the rest of the world.

Progress Report:

The original goals in the CIRM application were to use molecular DNA shuffling to make an AAV capsid library and then use the library to select for AAV vectors that would efficiently transduce human embryonic stem cells. We spent a long time preparing the capsid library and proving that our library had diverse sequences. In the end, we made two libraries each with about 100,000 variant capsids. We did our first set of screens on cultured cell lines and pulled out several novel AAV capsids. We vectorized them and showed they were much more efficient at transducing cells in culture than any other serotype described to date. There is a large amount of data describing the experiments in detail. These studies were published in a very long article published in the Journal of Virology in mid 2008 (Grimm et al., 2008.. In Vitro and In Vivo Gene Therapy Vector Evolution via Multispecies
Interbreeding and Retargeting of Adeno-Associated Viruses . J. Virol. 82:5887-5911

We had started using the capsid library in human embryonic stem cells as described. We had some technical problems with titering the right amount of human adenovirus needed as a helper virus in the designed screen. Unfortunately, time ran out and the grant ended and a new proposal to continue the work was not selected for review by CIRM. Nevertheless, since we believe producing the reagents we proposed to make for human ES stem cells are important for the research community and now that we have two very robust and diverse libraries, we are going to attempt another round of capsid screening. If we obtain useful serotypes for human ES cell transduction, we will make them available to the research community.

We should point out that we have recently produced DNA minicircle vectors, which are episomal plasmids that lack all bacterial plasmid backbone DNA. These minicircles have been used in collaboration with Joesph Wu and Michael Longaker to convert human adipose derived cells into iPS cells with 10 to 20 times greater efficiency than plasmid DNA. Moreover, these vectors are only transiently present in the iPS cells establishing that they do not permanently alter the genetic makeup of the cells. This work while not funded by the CIRM may turn out to be a more valuable approach for reprogramming all types of stem cells. If it were not for CIRM funding, this collaboration may not have occurred. The CIRM funding brought my group together with Joseph WU and Michael Longaker.